Emerging CRISPR Technologies

The discovery and implementation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) systems for genome editing has revolutionized biomedical research and holds great promise for the treatment of human genetic disorders. In addition to the popular CRISPR-Cas9 and CRISPR-Cpf1 systems for genome editing, several additional Class I and Class 2 CRISPR-Cas effectors have been identified and adapted for genome editing and transcriptome modulation. Here we discuss current and emerging CRISPR-based technologies such as Cascade-Cas3, CRISPR-associated transposases (CAST), CRISPR-Cas7–11, and CRISPR-Cas13 for genome and transcriptome modification. These technologies allow for the removal or insertion of large DNA elements, the modulation of gene expression at the transcriptional level, and the editing of RNA transcripts, expanding the capabilities of current technologies

Generation of Casp8FL122/123GG Mice Using CRISPR-Cas9 Technology

The purpose of this protocol is to describe the generation of missense mutations in mice using CRISPR-Cas9 technology. The current protocol focuses on the generation of a Casp8 FL122/123GG missense mutation, but it can be adapted to introduce any missense or nonsense mutation. For complete details on the use and execution of this protocol, please refer to Tummers et al. (2020)

SCYL1 disease and liver transplantation diagnosed by reanalysis of exome sequencing and deletion/duplication analysis of SCYL1

SCYL1 disease results from biallelic pathogenic variants in SCYL1. We report two new patients with severe hepatic phenotype requiring liver transplantation. Patient charts reviewed. DNA samples and skin fibroblasts were utilized. Literature was reviewed. 13-year-old boy and 9-year-old girl siblings had acute liver insufficiency and underwent living related donor liver transplantation in infancy with no genetic diagnosis. Both had tremor, global developmental delay, and cognitive dysfunction during their follow-up in the medical genetic clinic for diagnostic investigations after their liver transplantation. Exome sequencing identified a likely pathogenic variant (c.399delC; p.Asn133Lysfs*136) in SCYL1. Deletion/duplication analysis of SCYL1 identified deletions of exons 7–8 in Patient 1. Both variants were confirmed in Patient 2 and the diagnosis of SCYL1 disease was confirmed in both patients at the age of 13 and 9 years, respectively. SCYL1 protein was not expressed in both patients' fibroblast using western blot analysis. Sixteen patients with SCYL1 disease reported in the literature. Liver phenotype (n = 16), neurological phenotype (n = 13) and skeletal phenotype (n = 11) were present. Both siblings required liver transplantation in infancy and had variable phenotypes. Exome sequencing may miss the diagnosis and phenotyping of patients can help to diagnose patients

Gitelman-Like Syndrome Caused by Pathogenic Variants in mtDNA

Background: Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl− cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants of CLCNKB, HNF1B, FXYD2, or KCNJ10 may result in the same renal phenotype of Gitelman syndrome, as they can lead to reduced NCC activity. For approximately 10 percent of patients with a Gitelman syndrome phenotype, the genotype is unknown. Methods: We identified mitochondrial DNA (mtDNA) variants in three families with Gitelman-like electrolyte abnormalities, then investigated 156 families for variants in MT-TI and MT-TF, which encode the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. Mitochondrial dysfunction was induced in NCC-expressing HEK293 cells to assess the effect on thiazide-sensitive 22Na+ transport. Results: Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF), and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. All variants were classified as pathogenic, except for m.643A>G, which was classified as a variant of uncertain significance. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Dysfunction of oxidative phosphorylation complex IV and reduced maximal mitochondrial respiratory capacity were found in patient fibroblasts. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, inhibited NCC phosphorylation and NCC-mediated sodium uptake. Conclusion: Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies

Long-term outcomes of CLIPPERS (chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids) in a consecutive series of 12 patients.

BACKGROUND: Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a central nervous system inflammatory disease. OBJECTIVE: To describe the disease course of CLIPPERS. DESIGN: A nationwide study was implemented to collect clinical, magnetic resonance imaging, cerebrospinal fluid, and brain biopsy specimen characteristics of patients with CLIPPERS. SETTING: Academic research. PATIENTS: Twelve patients with CLIPPERS. MAIN OUTCOME MEASURES: The therapeutic management of CLIPPERS was evaluated. RESULTS: Among 12 patients, 42 relapses were analyzed. Relapses lasted a mean duration of 2.5 months, manifested frequent cerebellar ataxia and diplopia, and were associated with a mean Expanded Disability Status Scale (EDSS) score of 4. Besides typical findings of CLIPPERS, magnetic resonance imaging showed brainstem mass effect in 5 patients, extensive myelitis in 3 patients, and closed ring enhancement in 1 patient. Inconstant oligoclonal bands were found on cerebrospinal fluid investigation in 4 patients, with an increased T-cell ratio of CD4 to CD8. Among 7 available brain biopsy specimens, staining was positive for perivascular CD4 T lymphocytes in 5 samples. Thirty-eight of 42 relapses were treated with pulse corticosteroid therapy, which led to improvement, with a mean residual EDSS score of 1.9 (range, 0-7). In 1 patient with untreated relapses, scores on the EDSS progressively increased to a score of 10 at death. Among 5 patients without long-term corticosteroid therapy, the mean annualized relapse rate was 0.5 (range, 0.25-2.8). Among 7 patients taking oral corticosteroids, no relapses occurred in those whose daily dose was 20 mg or higher. No progressive course of CLIPPERS was observed. Four patients with a final EDSS score of 4 or higher had experienced previous severe relapses (EDSS score, ≥5) and brainstem and spinal cord atrophy. CONCLUSIONS: CLIPPERS is a relapsing-remitting disorder without progressive forms. Long-term disability is correlated with the severity of previous relapses. Further studies are needed to confirm that prolonged corticosteroid therapy prevents further relapses.journal article2012 Julimporte

SCYL pseudokinases in neuronal function and survival

The generation of mice lacking SCYL1 or SCYL2 and the identification of Scyl1 as the causative gene in the motor neuron disease mouse model muscle deficient (Scyl1 mdf/mdf) demonstrated the importance of the SCY1-like family of protein pseudokinases in neuronal function and survival. Several essential cellular processes such as intracellular trafficking and nuclear tRNA export are thought to be regulated by SCYL proteins. However, whether deregulation of these processes contributes to the neurodegenerative processes associated with the loss of SCYL proteins is still unclear. Here, I briefly review the evidence supporting that SCYL proteins play a role in these processes and discuss their possible involvement in the neuronal functions of SCYL proteins. I also propose ways to determine the importance of these pathways for the functions of SCYL proteins in vivo

CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

In this protocol, we describe the generation of conditional alleles in mice using the DECAI (DEgradation based on Cre-regulated Artificial Intron) approach. We detail steps for the CRISPR-mediated insertion of the short DECAI cassette within exon 3 of Scyl1 and the functional validation of alleles at genomic, transcriptomic, and protein levels. This strategy simplifies the process of generating mice with conditional alleles

Novel methods for the generation of genetically engineered animal models

Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology

One-step generation of a conditional allele in mice using a short artificial intron

Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice